Resistance in plants to infection by ssDNA virus using inoviridae virus ssDNA-binding protein, compositions and methods of use

ABSTRACT

The invention describes methods for producing plant resistance to a ssDNA virus, particularly a geminivirus such as mastrevirus, curtovirus or begomovirus. The method comprises introducing a ssDNA-binding protein of the  Inoviridae  virus into the plant, and includes a phage coat protein, particularly, a coliphage gene 5 protein. The invention also describes a transgenic plant comprising a gene that expresses the ssDNA-binding protein and vectors for expressing the protein in plants.

This application is a 371 of PCT/US99/04716, filed Mar. 3, 1999, whichclaims the benefit of U.S. Provisional Application No. 60/076,627, filedMar. 3, 1998, now abandoned.

This invention was made with government support under Contract No.2630152-1-3036-00 by the Agency for International Development. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The invention relates methods and compositions for producing plantswhich are resistant to infection by plant viruses.

BACKGROUND

Geminiviruses are plant pathogens that cause significant yield losses incrop plants in many countries of the world (Briddon et al,“Geminiviridae”, p. 158-165. In F. A. Murphy (ed.), Virus Taxonomy,Sixth Report of International Committee on Taxonomy of Viruses,Springer-Verlag, Vienna & New York, 1995; Frischmuth et al, Semin.Virol., 4:329-337, 1993; Harrison et al, Ann. Rev. Phytopathol.,23:55-82, 1985; Polston et al, Plant Dis., 81:1358-1369, 1997).Different members are transmitted by whiteflies or leafhoppers (Davieset al, Genet., 5:77-81, 1989; Lazarowitz et al, Crit. Rev. Plant Sci.,11:327-349, 1992). Most of the whitefly-transmitted geminiviruses (WTGs)have bipartite genomes while all the leafhopper-transmittedgeminiviruses and some of the WTGs have monopartite genomes. Themonopartite genomes (2566-3028 nt) encode proteins required forreplication, encapsidation and movement, while in the case of thebipartite viruses the movement functions are encoded by a second genomecomponent of similar size (Davies et al, Genet., 5:77-81, 1989; Inghamet al, Virology, 207:191-204, 1995; Timmermans et al, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 45:79-112, 1994).

Geminiviruses have circular single-stranded (ss) DNA genomesencapsidated in double icosahedral particles. Geminiviruses replicatevia a rolling circle mechanism analogous to replication ofbacteriophages with ssDNA genomes. The incoming geminivirussingle-stranded (ss) DNA is converted by host enzymes to double-stranded(ds) DNA which in turn serves as a template for transcription of early,replication associated genes on the complementary-sense strand.Replication initiator protein (Rep or AC1) is the only viral proteinrequired for replication. In bipartite geminiviruses, a second protein(AC3) enhances replication. AC2, another early gene product,transactivates expression of the coat protein (CP) gene on thevirion-sense strand. While the CP is not required for replication of thevirus in protoplasts or plants, mutations in CP lead to dramaticdecreases in accumulation of ssDNA in protoplasts or plants withoutaffecting the accumulation of dsDNA. On the other hand, tomato goldenmosaic virus CP mutations had no effect on DNA accumulation in plants,but reduced ssDNA accumulation while increasing the dsDNA accumulationin protoplasts. In plants, lack of CP results in a complete loss ofinfectivity of monopartite viruses but not bipartite viruses.

Coat protein may influence the ratios of ss and dsDNA levels in apassive manner by depleting the ssDNA that is available for conversionto dsDNA through encapsidation, or by modulating ssDNA synthesis, orboth. No evidence is available for how CP influences ssDNA accumulationin geminiviruses. In tomato leaf curl virus from New Delhi (ToLCV-NbE,hereafter referred as ToLCV), a geminivirus with bipartite genome,disrupting the synthesis of wild type CP resulted in drastic reductionin ssDNA and a three to five fold increase in dsDNA accumulation ininfected protoplasts. Inoculated plants, however, develop severesymptoms and accumulate wild type levels of dsDNA and low levels ofssDNA.

There remains a need to better understand the role of CP in geminivirusreplication.

BRIEF SUMMARY OF THE INVENTION

We have now discovered that a heterologous ssDNA binding protein cancomplement CP function in geminivirus ssDNA accumulation. It is alsodiscovered that ToLCV modified to express the ssDNA binding gene 5protein (g5p) from E. coli phage M13 in place of CP accumulates ssDNA towild type levels in protoplasts, but fails to move efficiently inplants, providing key insight into the present invention. Exemplaryheterologous ssDNA-binding proteins are found in the Inoviridae virusfamily.

Thus, in one embodiment, the invention describes a method for producingin a plant resistance to a single stranded DNA (ssDNA) virus comprisingintroducing a ssDNA-binding protein of the Inoviridae virus family intothe plant. The Inoviridae family virus ssDNA-binding protein is selectedfrom the group consisting of the Inovirus and Plectrovirus genuses, andthe Inovirus genus virus is selected from the group consisting ofColiphage, enterobacteria phage, Pseudomonas phage, Vibrio phage andXanthomonas phage species. A preferred Coliphage species of virus isselected from the group consisting of AE2, dA, Ec9, f1, fd, HR, M13,ZG/2 and ZJ/2 coliphages, with a coat protein or a gene 5 protein beingmore preferred. Particularly preferred is the Coliphage M13 gene 5protein.

The method of introduction of the ssDNA-binding protein into the plantcan include producing a transgenic plant containing an expression vectorfor expressing the protein, contacting a plant with an expression vectorfor expressing the protein, infecting the plant with a carrier vector,such as an Agrobacterium vector, and the like methods.

The invention also describes a DNA expression vector comprising anucleotide sequence that encodes a ssDNA-binding protein of theInoviridae virus family, wherein the vector is capable of expressing theprotein in plants. The vector is used in the methods described herein.

Also described is a composition for producing resistance to a ssDNAvirus that infects plants comprising an effective amount of a DNAexpression vector comprising a nucleotide sequence that encodes assDNA-binding protein of the Inoviridae virus family, wherein the vectoris capable of expressing the protein in the plant. In preferredembodiments, the vector is a carrier vector which can infect the plant.A particularly preferred vector is an Agrobacterium vector.

The invention also contemplates a transgenic plant containing a DNAexpression vector of this invention, which is resistant to ssDNA virusinfection due to the expression of a ssDNA binding protein as describedherein.

Other embodiments will be apparent from the teachings of thespecification and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the genome organization and schematic representationof constructs of tomato leaf curl virus from New Delhi (ToLCV-Nde). FIG.1A illustrates the genome organization of ToLCV-Nde showing the ORFs andtheir functions. CR, common region for both components. FIG. 1Billustrates a linear physical map of AV2 and CP region of ToLCV-Nde isshown at the bottom with nucleotide positions and relevant restrictionenzyme sites. The positions of different gene replacements are shownabove the linear map. Note that the gene replacements shown are not tothe scale. Descriptions of the constructs are given in Table 1.

FIG. 2 illustrates replication of ToLCV constructs in infected BY2protoplasts. Southern blot analysis was performed as described in theExamples. The viral constructs used for infecting protoplasts are shownabove the lanes. Protoplasts were inoculated with A component DNA alone(lanes 1-11) or coinoculated with A and B component DNAs (lanes 12-15).Each lane contained 4 μg of DNA prepared from protoplasts (singletransfection). Viral DNA was detected using a radioactively-labeledprobe from A component DNA. The position of supercoiled (sc),single-stranded (ss), open circular (op), and linear (li) viral DNAforms are indicated. Note that the positions of supercoiled and otherviral DNA forms in lane 11 are shifted upwards due to larger size of theCP66:6G:BC1 construct.

FIG. 3 illustrates indirect immunofluorescence of proteins expressed inprotoplasts (FIGS. 3A-3G) and fluorescence of green fluorescent protein(GFP) expressed in plants (FIGS. 3H-3P). Protoplasts were transfectedand antigens were visualized with different antibodies and FITC- orrhodamine-conjugated secondary antibody. GFP fluorescence in plants wasmonitored every three days for 15 days and the area shown corresponds to2.5×2.5 mm of leaf area. (FIG. 3A) Protoplast infected withCP66:Stag:6G:g5 virus and stained with S-protein coupled to FITC. (FIG.3B) Protoplast infected with wild type virus and stained with anti-CPantisera. (FIG. 3C) Protoplast infected with CP66:GUS virus and stainedwith anti-GUS antisera. (FIG. 3D) Protoplast infected with g5:GUSAV2⁻CP⁻virus and stained with anti-GUS antisera. (FIG. 3E) Protoplast infectedwith GUSAV2⁻CP⁻ virus and stained with anti-GUS antisera. (FIG. 3F)Protoplast infected with FBV1AV2⁻CP⁻ virus and stained with anti-Flagantibody. (FIG. 3G) Protoplasts infected with TBC1AV2⁻CP⁻ virus andstained with anti-T7 tag antibody. Note that two cells are shown in thismicrograph. Inoculated leaf (FIG. 3H) and systemic leaf (FIG. 3I) of aplant infected with GFPAV2⁻CP⁻+CP66:g5⁻ viruses 6 days post inoculation(dpi). Inoculated leaf (FIG. 3J) and systemic leaf (FIG. 3K) of a plantinfected with GFPAV2⁻CP⁻+CP66:g5⁻ viruses 15 dpi. Inoculated leaf (FIG.3L) and systemic leaf (FIG. 3M) of a plant infected withGFPAV2⁻CP⁻+CP66:6G:g5 viruses 6 dpi. Inoculated leaf (FIG. 3N) andsystemic leaves (FIGS. 3O and 3P) of a plant infected with GFPAV2⁻CP⁻+CP66:6G:g5 viruses 15 dpi.

FIG. 4 illustrates in vivo binding of gene 5 protein to ToLCV-Nde DNA.(FIG. 4A) Flag epitope-tagged CP66:6G:g5 protein expressed inprotoplasts was immunoprecipitated with anti-Flag antibody coupled toagarose after lysing protoplasts in NP40 buffer containing differentconcentrations of NaCl (shown above the lanes) or RIPA buffer, and theimmunoprecipitated protein was detected on a western blot with anti-Flagantibody (lanes 2-6). Lane 1 contained proteins immunoprecipitated fromprotoplasts transfected with wild type virus as a control. The proteinband present in all lanes at −24 kDa is the light chain of anti-Flagantibody used for immunoprecipitations. The immunoprecipitatedCP66:6G:g5 protein was detected at two different molecular massescorresponding to monomer and dimer forms. Positions of molecular weightmarkers are indicated in kilodaltons on the left. (FIG. 4B) Viral ssDNAthat coimmunoprecipitated with the Flag epitope-tagged CP66:6G:g5protein was detected on a Southern blot using 32P-labeled A componentDNA as a probe. Lanes 1-7 have same treatments as shown in FIG. 4A.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery that ssDNA-binding protein ofthe Inoviridae family of viruses interferes with virus spread during theinfection process of plant viruses of the ssDNA type. By inhibitingvirus spread, the virus infection is reduced and/or blocked, therebyincreasing plant “resistance” to the virus infection.

The invention describes methods for inhibiting ssDNA plant viruses usingInoviridae family virus ssDNA binding protein, expression vectorscapable of expressing the binding protein in plants, compositions fordelivery of the expression vectors, and transgenic plants containinggenes capable of expressing the binding protein.

A. Methods for Inhibiting ssDNA Plant Viruses

The invention contemplates methods for producing in a plant resistanceto infection and/or virulence of a single stranded DNA (ssDNA) virus.The method comprises introducing a ssDNA-binding protein from theInoviridae family virus into a susceptible plant.

The ssDNA-binding protein is typically provided by expression of anucleotide sequence which encodes the ssDNA-binding protein and whichcontains expression control elements which provide for expression ofprotein in plants.

Introduction of the protein into the plant can be accomplished by avariety of methods including standard gene transfer methods,innoculation of the plant with a transfer or carrier vector (i.e.,infection by an engineered plant virus or phage), “biolistic” (i.e.,ballistic) introduction of nucleic acids into mature plant tissue,direct DNA uptake into plant protoplast, transformation of plants withAgrobacterium tumefaciens-based vectors, and the like well knownmethods.

Plant expression elements for a nucleotide sequence are generally wellknown in the art and are not to be considered limiting to the invention.The nucleotide sequence which encodes the ssDNA-binding protein can bepresent on an expression vector, as a DNA fragment, or as a component ofa “transfer” or carrier vector such as the infectious Agrobacterium genetransfer system commonly used in plants.

A preferred ssDNA-binding protein is an Inoviridae family virus proteinhaving the ability to bind ssDNA. The preferred protein is either theviral coat protein or the viral “gene 5” protein. Although whole (i.e.,native) protein can be used, portions of the whole protein can also beused that contain the ssDNA binding portion of the protein. In addition,it is understood that modifications to the amino acid residue sequenceof a native protein can be made without compromising the essentialfunctional properties of the protein according to the invention. Thus,the term “ssDNA-binding protein” means any of a variety of configurationof protein including active fragments, fusion proteins containing anactive fragment, whole protein, and derivatives thereof which possessthe ssDNA binding activity.

The ability to bind ssDNA can be readily measured by art-recognizedprocedures, including the binding methods described herein. Thus, theinvention is not to be construed as so limited so long as thessDNA-binding protein has the ability to bind plant viral ssDNA asdescribed herein, and inhibit virus replication and/or viralpathogenesis.

The Inoviridae family of viruses is a large family that includes theInovirus and Plectrovirus genera. Preferred Inovirus species includeColiphage, enterobacteria phage, Pseudomonas phage, Vibrio phage andXanthomonas phage species. Preferred Coliphage species include AE2, dA,Ec9, f1, fd, HR, M13, ZG/2 and ZJ/2 coliphages. A particularly preferredprotein is the Coliphage M13 gene 5 protein.

In preferred embodiments, the Coliphage M13 gene 5 protein has the aminoacid residue sequence shown in SEQ ID NO 1.

In a related embodiment, the method comprises introducing thessDNA-binding protein by preparing a transgenic plant which comprises agene capable of expressing the protein, and thereby providing plantresistance to the ssDNA plant virus. The methods for preparing atransgenic plant capable of expressing an foreign protein such as thessDNA-binding protein of this invention are described further herein.

In a further related embodiment, the methods comprises introducing thessDNA-binding protein by contacting the plant with a compositioncontaining an expression vector capable of expressing the protein in theplant. Methods for preparing and using an expression vector in acomposition according to the invention are described further herein.

In the various nucleic acid-based methods in which a nucleotide sequenceencodes the ssDNA-binding protein and is capable of expressing theprotein, it is understood that the nucleotide sequence can vary incontent so long a contemplated ssDNA-binding protein is encoded. Forexample, the genetic code tolerates variation in codon usage forencoding an amino acid residue sequence, and therefore the invention isnot to be construed as limited to a particular nucleotide sequence.However, it is also understood that an expression environment, e.g., theplant cell, has codon usage preferences, and therefore it is desirableto utilize the preferred codons to optimize expression of expressiblegenes in plants.

In this regard, a preferred nucleotide sequence for use in an expressionvector or transgenic plant of this invention can utilize preferredcodons. A particularly preferred nucleotide sequence for use in thepresent invention encodes an M13 gene 5 protein, preferably the aminoacid residue sequence shown in SEQ ID NO 1. In one embodiment, apreferred nucleotide sequence comprises the nucleotide sequence shown inSEQ ID NO 2 which is the native nucleotide sequence from the M13 viralgenome encoding the native M13 gene 5 protein. In another embodiment, apreferred nucleotide sequence comprises the nucleotide sequence shown inSEQ ID NO 3 which is a synthetic nucleotide sequence designed toincorporate preferred codon usages for highly expressed human genes, andwhich encodes the native M13 gene 5 protein.

The complete sequence of bacteriophage M13, including the gene 5 codingsequence, is available from GenBank as Accession numbers V00604, J02461and M10377. The amino acid residue sequence and nucleotide sequenceencoding M13 gene 5 is shown in SEQ ID Nos 1 and 2, respectively.

The introduced protein is effective at inhibiting infection of any ssDNAvirus that infects plants. Preferred viruses are the Geminiviridaefamily of viruses, which includes Mastrevirus, Curtovirus andBegomovirus genera.

Preferred Mastrevirus genus species are selected from the groupconsisting of Bajra streak virus, Bean yellow dwarf virus, Bromusstriate mosaic virus, Chickpea chlorotic dwarf virus, Chloris striatemosaic virus, Digitaria streak virus, Digitaria striate mosaic virus,Maize streak virus//Ethiopia, Maize streak virus//Ghanal, Maize streakvirus//Ghana2, Maize streak virus//Kenya, Maize streakvirus//Komatipoort, Maize streak virus//Malawi, Maize streakvirus//Mauritius, Maize streak virus//Mozambique, Maize streakvirus//Nigeria1, Maize streak virus//Nigeria2, Maize streakvirus//Nigeria3, Maize streak virus//Port Elizabeth, Maize streakvirus//Reunion1, Maize streak virus//Reunion2, Maize streakvirus//Setaria, Maize streak virus//South Africa, Maize streakvirus//Tas, Maize streak virus//Uganda, Maize streak virus//Vaalhartmaize, Maize streak virus//Vaalhart wheat, Maize streakvirus//Wheat-eleusian, Maize streak virus//Zaire, Maize streakvirus//Zimbabwel, Maize streak virus//Zimbabwe2, Miscanthus streakvirus, Panicum streak virus/Karino, Panicum streak virus/Kenya, Paspalumstriate mosaic virus, Sugarcane streak virus//Egypt, Sugarcane streakvirus/Natal, Sugarcane streak virus/Mauritius, Tobacco yellow dwarfvirus, Wheat dwarf virus/Czech Republic [Wheat dwarf virus-CJI,WDV-CJI], Wheat dwarf virus/France and Wheat dwarf virus/Sweden.

Preferred Curtovirus genus species are selected from the groupconsisting of Beet curly top virus-California, Beet curly topvirus-California//Logan, Beet curly top virus-CFH, Beet curly topvirus//Iran, Beet curly top virus-Worland, Horseradish curly top virus,Tomato leafroll virus and Tomato pseudo-curly top virus.

Preferred Begomovirus genus species are selected from the groupconsisting of Abutilon mosaic virus, Acalypha yellow mosaic virus,African cassava mosaic virus//Ghana, African cassaya mosaic virus/Kenya,African cassaya mosaic virus/Nigeria, African cassaya mosaicvirus/Uganda, Ageratum yellow vein virus, Althea rosea enation virus,Asystasia golden mosaic virus, Bean calico mosaic virus, Bean dwarfmosaic virus, Bean golden mosaic virus-Brazil, Bean golden mosaicvirus-Puerto Rico, Bean golden mosaic virus-Puerto Rico/Dominican Rep.[Bean golden mosaic virus-Dominican Rep., BGMV-DR], Bean golden mosaicvirus-Puerto Rico/Guatemala [Bean golden mosaic virus-Guatemala,BGMV-GA], Bhendi yellow vein mosaic virus, Chino del tomate virus[Tomato leaf crumple virus, TLCrV], Cotton leaf crumple virus, Cottonleaf curl virus-India, Cotton leaf curl virus-Pakistan1/Faisalabad1[Cotton leaf curl virus-Pakistan2], Cotton leaf curlvirus-Pakistan1/Faisalabad2 [Cotton leaf curl virus-Pakistan3], Cottonleaf curl virus-Pakistan1/Multan [Cotton leaf curl virus-Pakistan1],Cotton leaf curl virus-Pakistan2/Faisalabad [Pakistani cotton leaf curlvirus], Cowpea golden mosaic virus, Croton yellow vein mosaicvirus//Lucknow, Dolichos yellow mosaic virus, East african cassayamosaic virus/Kenya, East african cassaya mosaic virus/Malawi, Eastafrican cassaya mosaic virus/Tanzania, East african cassaya mosaicvirus/Uganda//1 [African cassaya mosaic virus-Uganda variant], Eastafrican cassaya mosaic virus/Uganda//2, Eclipta yellow vein virus,Eggplant yellow mosaic virus, Eupatorium yellow vein virus, Euphorbiamosaic virus, Honeysuckle yellow vein mosaic virus, Horsegram yellowmosaic virus, Indian cassaya mosaic virus, Jatropha mosaic virus,Leonurus mosaic virus, Limabean golden mosaic virus, Lupin leaf curlvirus, Macroptilium golden mosaic virus-Jamaica//2, Macroptilium goldenmosaic virus-Jamaica//3, Macrotyloma mosaic virus, Malvaceous chlorosisvirus, Melon leaf curl virus, Mungbean yellow mosaic virus, Okra leafcurl virus//Ivory Coast, Okra leaf curl virus//India, Papaya leaf curlvirus, Pepper huasteco virus, Pepper golden mosaic virus, [Texas peppervirus], Pepper mild tigrA virus, Potato yellow mosaic virus//Guadeloupe,Potato yellow mosaic virus/Trinidad and Tobago, Potato yellow mosaicvirus/Venezuela, Pseuderanthemum yellow vein virus, Rhynchosia mosaicvirus, Serrano golden mosaic virus, Sida golden mosaic virus/Costa Rica,Sida golden mosaic virus/Honduras, Sida golden mosaicvirus/Honduras//Yellow vein, Sida yellow vein virus, Solanum apical leafcurl virus, Soybean crinkle leaf virus, Squash leaf curl virus, Squashleaf curl virus/Extended host, Squash leaf curl virus/Restricted host,Squash leaf curl virus/Los Mochis, Squash leaf curl virus-China, Tomatogolden mosaic virus/Common strain, Tomato golden mosaic virus/Yellowvein strain, Tobacco leaf curl virus//India, Tobacco leaf curlvirus-China, Tomato leaf curl virus//Solanum species D1, Tomato leafcurl virus//Solanum species D2, Tomato leaf curl virus-Australia, Tomatoleaf curl virus-Bangalore1 [Indian tomato leaf curl virus-BangaloreI],Tomato leaf curl virus-Bangalore2, [Indian tomato leaf curl virus,ItoLCV], Tomato leaf curl virus-Bangalore3 [Indian tomato leaf curlvirus-BangaloreII], Tomato leaf curl virus-New Delhi/Severe [Tomato leafcurl virus-India2, ToLCV-IN1], Tomato leaf curl virus-New Delhi/Mild[Tomato leaf curl virus-India2, ToLCV-IN2], Tomato leaf curl virus-NewDelhi/Lucknow [Indian tomato leaf curl virus], Tomato leaf curlvirus//Senegal, Tomato leaf curl virus-Sinaloa [Sinaloa tomato leaf curlvirus, STLCV], Tomato leaf curl virus-Taiwan, Tomato leaf curlvirus-Tanzania, Tomato mottle virus, Tomato mottle virus-Taino [Tainotomato mottle virus, TTMoV], Tomato severe leaf curl virus//Guatemala,Tomato severe leaf curl virus//Honduras, Tomato severe leaf curlvirus//Nicaragua, Tomato yellow dwarf virus, Tomato yellow leaf curlvirus-China, Tomato yellow leaf curl virus-Israel, Tomato yellow leafcurl virus-Israel/Mild, Tomato yellow leaf curl virus-Israel/Egypt,[Tomato yellow leaf curl virus-Egypt, TYLCV-EG], Tomato yellow leaf curlvirus-Israel//Cuba, Tomato yellow leaf curl virus-Israel//Jamaica,Tomato yellow leaf curl virus-Israel//Saudi Arabia1, [Tomato yellow leafcurl virus-Northern Saudi Arabia, TYLCV-NSA], Tomato yellow leaf curlvirus-Nigeria, Tomato yellow leaf curl virus-Sardinia, Tomato yellowleaf curl, virus-Sardinia/Sicily [Tomato yellow leaf curl virus-Sicily,TYLCV-SY], Tomato yellow leaf curl virus-Sardinia/Spain//1[Tomato yellowleaf curl virus-Spain, TYLCV-Sp], Tomato yellow leaf curlvirus-Sardinia/Spain//2 [Tomato yellow leaf curl virus-Almeria,TYLCV-Almeria], Tomato yellow leaf curl virus-Sardinia/Spain//3 [Tomatoyellow leaf curl virus-European strain], Tomato yellow leaf curlvirus-Saudi Arabia [Tomato yellow leaf curl virus-Southern Saudi Arabia,TYLCV-SSA], Tomato yellow leaf curl virus-Tanzania, Tomato yellow leafcurl virus-Thailand//1, Tomato yellow leaf curl virus-Thailand//2,Tomato yellow leaf curl virus//Yemen, Tomato yellow mosaicvirus-Brazil//1, Tomato yellow mosaic virus-Brazil//2, Tomato yellowmottle virus, Tomato yellow vein streak virus-Brazil, Watermelonchlorotic stunt virus, Watermelon curly mottle virus and Wissadulagolden mosaic virus-Jamaica//1.

Other ssDNA plant viruses include Banana bunchy top virus, Coconutfoliar decay virus, Fababean necrotic yellows virus, Milk vetch dwarfvirus and Subterranean clover stunt virus.

The above described ssDNA plant viruses which can be inhibited by thepresent methods infect a large number of plant species. Insofar as newplant species can be discovered which are susceptible to infection by assDNA plant virus described according to the present invention, it is tobe understood that the invention is not intended to be so limited toknown plants. Instead, a plant according to the present methods isintended to be any plant which is susceptible to infection by thedescribed ssDNA plant virus, which susceptibility can be readilydetermined by art recognized methods, including the infection proceduresdescribed herein.

The term “plant” includes whole plants, plant organs (e.g., leaves,stems, roots, etc.), seeds and plant cells and progeny of same. Theclass of plants which can be used in the method of the invention isgenerally as broad as the class of higher plants amenable totransformation techniques, including both monocotyledonous (monocots)and dicotyledonous (dicots) plants. It includes plants of a variety ofploidy levels, including polyploid, diploid and haploid.

Exemplary plants which are susceptible to infection, and therefore aretargets for the treatment methods and compositions described hereininclude, but are not limited to, a plant is selected from the groupconsisting of Abutilon, Acalypha, apple, Ageratum, Althea rosea,Asystasia, Bajra, banana, barley, beans, beet, Blackgram, Bromus,Cassaya, chickpea, Chilllies, Chloris, clover, coconut, coffee, cotton,cowpea, Croton, cucumber, Digitaria, Dolichos, eggplant, Eupatorium,Euphorbia, fababean, honeysuckle, horsegram, Jatropha, Leonurus,limabean, Lupin, Macroptilium, Macrotyloma, maize, melon, millet,mungbean, oat, okra, Panicum, papaya, Paspalum, peanut, pea, pepper,pigeon pea, pineapple, Phaseolus, potato, Pseuderanthemum, pumpkin,Rhynchosia, rice, Serrano, Sida, sorghum., soybean, squash, sugarcane,sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon,wheat and Wissadula, or any individual plant or combination of plantsthereof.

Preferred examples of the methods of the invention are described hereinusing the M13 gene 5 protein expressed using a recombinant tomato leafcurl virus (ToLCV) vector on tobacco plants and protoplasts. The ToLCVviral genomic nucleotide sequences for both the A and B components ofthe ToLCV bipartite genome are known, and are available as GenBankAccession numbers U15015 and U15016, respectively, and are shown in SEQID NOs 4 and 5, respectively.

B. Nucleic Acid Molecules

The invention also contemplates a nucleic acid molecule, such as a DNAexpression vector, useful for expression of a ssDNA-binding protein ofthis invention in plants. Thus the nucleic acid molecule contains anucleotide sequence which encodes the ssDNA-binding protein of thisinvention and further contains elements for regulation and control ofgene expression in plants. Exemplary elements for expression in plantsare described in U.S. Pat. Nos. 5,188,642, 5,202,422, 5,463,175 and5,639,947, the disclosures of which are hereby incorporated byreference. In addition, the methods of manipulating nucleic acids andthe production of expression vectors for use in plants is generally wellknown and therefore not to be construed as limiting to the presentinvention.

Exemplary expression vectors and systems for introduction of assDNA-binding protein into plants are described in the Examples.

Thus, in one embodiment, the invention describes a nucleic acid-basedexpression system comprising a nucleotide sequence that encodes assDNA-binding protein of the Inoviridae virus family, where theexpression system is capable of expressing the protein in a plantsusceptible to infection by a ssDNA plant virus as described herein.

The sSDNA-binding protein can be any protein as described herein and asis preferred in practicing the methods for the invention. Particularlypreferred is the M13 gene 5 protein, such as the amino acid residuesequence shown in SEQ ID NO 1.

The expression system can be a vector or a gene, depending upon thecontemplated usage. In the case of a transgenic plant, the inventiondescribes a gene comprising a nucleotide sequence which defines anexpression cassette, i.e., the necessary elements for expression of assDNA-binding protein structural gene including promoters, transcriptionstart signals, translation start signals, the structural protein codingsequence, and translation and transcription stop sequences, as are wellknown. In the case of a vector or infectious agent used to introduce anexpression cassette, the vector or agent comprises additional geneticelements suitable for the vector or infectious agent's function.

For example, the vector may also contain elements which provide forreplication, manipulation and the like, such as in found on plasmidswhich facilitate bulk preparation of the vector. In the case ofinfectious agents, which are typically modified plant viruses or plantphage which can infect the plant, the agent may contain additionalelements for replication of the agent and assembly into an infectiousparticle, as are well known.

A preferred expression cassette in a vector, gene or infectious agentaccording to the invention comprises a nucleotide sequence shown in SEQID NOs 2 or 3 as described herein.

For general cloning of nucleic acids, plasmids are used as are wellknown. A preferred cloning plasmid used herein is the pBluescript II SKvector (Stratagene, La Jolla, Calif.). The complete nucleotide sequenceof the pBluescript plasmid is available in GenBank as Accession numberX52330, and is also shown in SEQ ID NO 6.

For plant transformations, a variety of methods, vectors and agents areavailable, and therefore the invention is not to be construed as solimited. Exemplary methods include plant transformation, comprisingdirect uptake of an expression cassette nucleic acid(s) into aprotoplast followed by plant regeneration to form a plant,electroporation into a protoplast, biolistic delivery of nucleic acidinto either cultured plant cells or whole plant tissue, pollen-mediatedtransformations, infection by a recombinant virus or phage agent, suchas the modified ToLCV or an Agrobacterium-mediated transformation, andthe like. Exemplary vectors for conducting some of the above methodsinclude pBIN19 (Bevan et al, Nucl. Acids Res., 12:8711, 1984; GenBankAccession number U09365), pMON316 or pMON available from Monsanto (St.Louis, Mo.), pGA482 (An et al, Plant Physiol., 81:86, 1986), pCGN1547(McBride et al, Plant Mol. Biol., 14:269, 1990), pPZP100 (Ajdukiewicz etal, Plant Mol. Biol., 25:989, 1994, and GenBank Accession numberU10456), pMOG410, and the like.

C. Transgenic Plants

The invention further contemplates a transgenic plant containing anucleotide sequence of this invention for expressing the ssDNA-bindingprotein. The transgenic plant contains an expression cassette as definedherein as a part of the plant, the cassette having been introduced bytransformation of a plant with a vector of this invention.

Methods for producing a transgenic plant useful in the present inventionare described in U.S. Pat. Nos. 5,188,642; 5,202,422; 5,234,834;5,463,175; and 5,639,947, the disclosures of which are herebyincorporated by reference.

Techniques for transforming a wide variety of plant species are alsowell known and described in the technical and scientific literature.See, for example, Weising et al, Ann. Rev. Genet., 22:421-477, 1988. Aconstitutive or inducible promoter is operably linked to the desiredheterologous DNA sequence encoding a ssDNA-binding protein of thisinvention in a suitable vector. The vector comprising a promoter fusedto the heterologous DNA will typically contain a marker gene whichconfers a selectable phenotype on plant cells. For example, the markermay encode biocide resistance, particularly antibiotic resistance, suchas resistance to kanamycin, G418, bleomycin, hygromycin, or herbicideresistance, such as resistance to chlorsulfuron or Basta. Such selectivemarker genes are useful in protocols for the production of transgenicplants.

DNA constructs containing the expression cassette can be introduced intothe genome of the desired plant host by a variety of conventionaltechniques. For example, the DNA construct may be introduced directlyinto the DNA of the plant cell using techniques such as electroporationand microinjection of plant cell protoplasts. Alternatively, the DNAconstructs can be introduced directly to plant tissue using biolisticmethods, such as DNA micro-particle bombardment. In addition, the DNAconstructs may be combined with suitable T-DNA flanking regions andintroduced into a conventional Agrobacterium tumefaciens host vector.The virulence functions of the Agrobacterium tumefaciens host willdirect the insertion of the construct and adjacent marker into the plantcell DNA when the cell is infected by the bacteria.

Microinjection techniques are known in the art and well described in thescientific and patent literature. The introduction of DNA constructsusing polyethylene glycol precipitation is described in Paszkowski etal, EMBO J., 3:2717-2722, 1984. Electroporation techniques are describedin Fromm et al, Proc. Natl. Acad. Sci. USA, 82:5824, 1985. Biolistictransformation techniques are described in Klein et al, Nature327:70-73, 1987. The full disclosures of all references cited areincorporated herein by reference.

A variation involves high velocity biolistic penetration by smallparticles with the nucleic acid either within the matrix of small beadsor particles, or on the surface (Klein et al, Nature, 327:70-73, 1987).Although typically only a single introduction of a new nucleic acidsegment is required, this method particularly provides for multipleintroductions.

Agrobacterium tumefaciens-meditated transformation techniques are welldescribed in the scientific literature. See, for example Horsch et al,Science, 233:496-498, 1984, and Fraley et al, Proc. Natl. Acad. Sci.USA, 90:4803, 1983. More specifically, a plant cell, an explant, ameristem or a seed is infected with Agrobacterium tumefacienstransformed with the segment. Under appropriate conditions known in theart, the transformed plant cells are grown to form shoots, roots, anddevelop further into plants. The nucleic acid segments can be introducedinto appropriate plant cells, for example, by means of the Ti plasmid ofAgrobacterium tumefaciens. The Ti plasmid is transmitted to plant cellsupon infection by Agrobacterium tumefaciens, and is stably integratedinto the plant genome (Horsch et al, Science, 233:496-498, 1984; Fraleyet al, Proc. Nat'l. Acad. Sci. U.S.A., 80:4803, 1983.

Ti plasmids contain two regions essential for the production oftransformed cells. One of these, named transfer DNA (T DNA), inducestumor formation. The other, termed virulent region, is essential for theintroduction of the T DNA into plants. The transfer DNA region, whichtransfers to the plant genome, can be increased in size by the insertionof the foreign nucleic acid sequence without its transferring abilitybeing affected. By removing the tumor-causing genes so that they nolonger interfere, the modified Ti plasmid can then be used as a vectorfor the transfer of the gene constructs of the invention into anappropriate plant cell, such being a “disabled Ti vector”.

All plant cells which can be transformed by Agrobacterium and wholeplants regenerated from the transformed cells can also be transformedaccording to the invention so as to produce transformed whole plantswhich contain the transferred foreign nucleic acid sequence.

There are various ways to transform plant cells with Agrobacterium,including:

-   -   (1) co-cultivation of Agrobacterium with cultured isolated        protoplasts,    -   (2) co-cultivation of cells or tissues with Agrobacterium, or    -   (3) transformation of seeds, apices or meristems with        Agrobacterium.

Method (1) requires an established culture system that allows culturingprotoplasts and plant regeneration from cultured protoplasts.

Method (2) requires (a) that the plant cells or tissues can betransformed by Agrobacterium and (b) that the transformed cells ortissues can be induced to regenerate into whole plants.

Method (3) requires micropropagation.

In the binary system, to have infection, two plasmids are needed: aT-DNA containing plasmid and a vir plasmid. Any one of a number of T-DNAcontaining plasmids can be used, the only requirement is that one beable to select independently for each of the two plasmids.

After transformation of the plant cell or plant, those plant cells orplants transformed by the Ti plasmid so that the desired DNA segment isintegrated can be selected by an appropriate phenotypic marker. Thesephenotypic markers include, but are not limited to, antibioticresistance, herbicide resistance or visual observation. Other phenotypicmarkers are known in the art and may be used in this invention.

The present invention embraces use of the expression vectors describedherein in transformation of any plant, including both dicots andmonocots. Transformation of dicots is described in references above.Transformation of monocots is known using various techniques includingelectroporation (e.g., Shimamoto et al, Nature, 338:274-276, 1992;ballistics (e.g., European Patent Application 270,356); andAgrobacterium (e.g., Bytebier et al, Proc. Nat'l Acad. Sci. USA,84:5345-5349, 1987).

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the desired transformed phenotype. Such regenerationtechniques rely on manipulation of certain phytohormones in a tissueculture growth medium typically relying on a biocide and/or herbicidemarker which has been introduced together with the nucleotide sequences.Plant regeneration from cultured protoplasts is described in Evans etal, Handbook of Plant Cell Culture, pp. 124-176, MacMillan PublishingCompany, New York, 1983; and Binding, Regeneration of Plants, PlantProtoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration canalso be obtained from plant callus, explants, organs, or parts thereof.Such regeneration techniques are described generally by Klee et al, Ann.Rev. Plant Phys., 38:467-486, 1987.

One of skill will recognize that, after an expression cassette is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

D. Compositions

Also contemplated is a composition useful for introducing a nucleotidesequence of this invention into plants. The composition is useful forproducing resistance to a ssDNA virus that infects plants, and comprisesan effective amount of the nucleotide sequence according to theinvention for introducing the ssDNA-binding protein into a plant, anddepends upon the method used for introducing the protein to the plant.For example, using direct DNA uptake by protoplast, the composition is aaqueous solution containing nucleic acid and buffers to facilitateuptake by protoplast, as is well known. For transformation by anAgrobacterium vector, the composition contains a suspension ofAgrobacteria containing the nucleotide sequence capable of expressingthe sSDNA-binding protein.

E. Systems for Use

The present invention also contemplates a system, preferably in kitform, useful for practicing the methods of the present invention. Thus,the kits are useful for introducing a nucleic acid sequence of thepresent invention into a plant as practiced in the methods of thisinvention.

The kit comprises, in an amount sufficient to perform at least oneintroduction, a composition of the present invention comprising anucleic acid molecules which comprise a nucleotide sequence capable ofexpressing a ssDNA-binding protein according to the present invention,present in a packaging material or container for providing the system.

Instructions for use of the packaged reagent are also typically includedin the system in the form of a label or packaging insert.

“Instructions for use” typically include a tangible expressiondescribing the contents of the reagent(s) in the system or at least onemethod parameter such as the relative amounts of composition and plantto be admixed, procedures for contacting the plant, temperature, bufferconditions and the like for practicing a method of the invention.Typically, the instructions will recite the method for contacting aplant to introduce the ssDNA-binding protein of the invention into aplant, and thereby inhibit symptoms of ssDNA virus infection in theplant.

The reagent species, infectious agent, virus or phage, nucleic acidmolecule or expression vector for practicing a method described hereincan be provided in solution, as a liquid dispersion or as asubstantially dry power, e.g., in lyophilized form.

The term “package” refers to a solid matrix or material such as glass,plastic (e.g., polyethylene, polypropylene and polycarbonate), paper,foil and the like capable of holding within fixed limits a reagent suchas a polynucleotide, transformation agent, infectious virus or phage ofthe present invention. Thus, for example, a package can be a bottle,vial, plastic and plastic-foil laminated envelope or the like containerused to contain a contemplated composition.

The package can contain one or more unit dosages of the composition ofthe invention, or may alternatively be packaged with the compositionprovided in bulk.

A system of this invention may comprise a carrier means beingcompartmentalized to receive in close confinement one or more containermeans such as vials, tubes, and the like, each of the container meanscomprising one of the separate elements to be used in the method. Forexample, one of the container means may comprise a composition forinfecting a plant. The kit may also have containers containing any otherreagents used to practice the methods of the invention.

Other uses will be apparent to one skilled in the art in light of thepresent disclosures and the examples that follow.

EXAMPLES

The following examples are provided by way of illustration and notlimitation.

1. Plasmid Constructs

Infectious clones of the A and B components of tomato leaf curl virus(Padidam et al, J. Gen. Virol., 76:25-35, 1995) were employed togenerate the virus constructs used herein. The genome organization ofToLCV and schematic representation of virus constructs used are shown inFIG. 1 and the detailed descriptions and methods of construction of eachof the plasmid are summarized in Table 1. Partial head to tail dimersmade from these constructs were used to infect Nicotiana benthamianaplants and N. tabacum BY2 protoplasts.

TABLE 1 Description and method of construction of viral DNAs ConstructDescription and method of construction AV2⁻CP⁻ A double mutant of AV2and coat protein (CP) in which Met1 codon of AV2 was changed totermination codon and Arg66 codon of CP was frame shifted. The mutanthas been described earlier as M1te/R66fr (Padidam et al, Virology,224:390-404, 1996) g5AV2⁻CP⁻ A 264-bp sequence coding for gene 5 (g5)protein from bacteriophage M13mp18 vector was amplified by PCR (10cycles) and cloned between Afl III (nt 125) and Sty I (nt 479) sitesresulting in replacement of AV2 ORF and overlapping 5′ CP ORF sequenceswith g5. g5⁻AV2⁻CP⁻ A negative control of g5AV2⁻CP⁻construct in whichMet1 codon of g5 was mutated to a termination codon. CP⁻ A mutant of CPmade by end-filling and religation at the unique Sty I site (nt 479)causing frame shift at Arg66 codon and termination after amino acid (aa)69. The mutant has been described earlier as R66fr (Padidam et al,Virology, 224:390-404, 1996). CP66:g5 A 264 bp sequence coding for g5protein from M13mp18 vector was amplified by PCR (10 cycles) and clonedbetween and Sty I (nt 479) and Sph I (nt 836) sites resulting in fusionof g5 sequence to Arg66 codon of CP. CP66:6G:g5 Similar to CP66:g5except that an oligonucleotide coding for 6 glycines was insertedbetween codons for Arg66 of CP and Met1 of g5. CP66:g5⁻ A negativecontrol in which Arg66 codon of CP66:g5 was frame shifted.CP66:Stag:6G:g5 Similar to CP66:6G:g5 except that a sequence coding forthe 15 aa Stag peptide epitope [KETAAAKFERQHMDS (SEQ ID NO:7); (Kim etal, J.S., Protein Sci., 2:348-356, 1993)] was inserted after Arg66 codonof CP. Stag epitope was inserted to immunolocalize the CP66:6G:g5protein in protoplasts using the S-protein coupled to the FITC.FCP66:6G:g5 A sequence coding for 9 aa Flag peptide epitope [MDYKDDDDK(SEQ ID NO:8); (Ropp et al, J. Immunol. Methods., 88:1-18, 1986)] wasadded before the Met1 codon of CP66:6G:g5 and cloned between Afl III (nt125) and Sph I (nt 836). AV2 ORF is deleted in this construct. Flagepitope was added to immunoprecipitate the CP66:6G:g5 protein fromprotoplasts using the anti-Flag antibody. CP66:GUS A 1806-bp DNAfragment coding for β-glucuronidase (GUS) protein (Jefferson et al,Plant Mol. Biol. Rep., 5:387-405, 1987) was PCR amplified (10 cycles)and cloned between Sty I (nt 479) and Hind III (nt 1041) sites of Acomponent. The Hind III site was created at the codon for Tyr251 of CP[15-bp before the termination codon, (Padidam et al, Virology,224:390-404, 1996)]. This facilitated replacement CP sequence with othersequences. GUSAV2⁻CP⁻ A 1869-bp Nco I to EcoR I DNA fragment coding forGUS protein was cloned between Afl III (nt 125) and Hind III (nt 1041)sites of A component after blunt ending the EcoR I site on the GUS geneand Hind III site on A component DNA. GFPAV2⁻CP⁻ A 717-bp with Nco I toBamH I DNA fragment coding for green fluorescent protein [GFP - S65C,M153T, V163A; (Reichel et al, Proc. Natl. Acad. Sci. USA, 93:5855-5893,1996)] was cloned between Afl III (nt 125) and Sph I (nt 836) sites of Acomponent after blunt ending the BamH I site on the GFP gene and Sph Isite on A component DNA. BV1AV2⁻CP⁻ A 849-bp sequence coding for BV1from B component of ToLCV was amplified by PCR (10 cycles) and clonedbetween Afl III (nt 125) and Hind III (nt 1041) sites of A component.FBV1AV2⁻CP⁻ Similar to BV1AV2⁻CP⁻ except that sequence coding for 9 aaFlag peptide was added before the Met1 codon of BV1. Flag epitope wasadded to immunolocalize the BV1 protein in protoplasts using theanti-Flag antibody. BC1AV2⁻CP⁻ A 882-bp sequence coding for BC1 from Bcomponent of ToLCV was amplified by PCR (10 cycles) and cloned betweenAfl III (nt 125) and Hind III (nt 1041) sites of A component.TBC1AV2⁻CP⁻ Similar to BC1AV2⁻CP⁻ except that sequence coding for 11 aaT7 [MASMTGGQQMG (SEQ ID NO:9); (Krek et al, Cell, 78:161-172, 1994)]epitope was added before the Met1 codon of BC1. T7 tag epitope was addedto immunolocalize the BC1 protein in protoplasts using the anti-T7 tagantibody. Cp66:6G:BC1 A 900-bp sequence coding for 6 glycines and BC1from B component of ToLCV was amplified by PCR (10 cycles) and clonedbetween Sty I (nt 479) and Hind III (nt 1041) sites. BC1⁻ B componentDNA in which a frame-shift mutation of BC1 was created by deleting the3′ overhang and religating at the Pst I site (nt 2075) Described earlieras BC1M (Padidam et al, Virology, 224:390-404, 1996)2. Protoplast and Plant Inoculations

N. benthamiana plants (two week-old seedlings grown in Magenta boxes)and protoplasts isolated from BY2 suspension cells were infected withviral DNAs as described earlier (Padidam et al, J. Gen. Virol.,76:25-35, 1995; Padidam et al, Virology, 224:390-404, 1996). Protoplastswere collected from cultures 48 h postinoculation for DNA isolation,immunoprecipitation reactions, and western blot analysis. Plants werescored for symptoms, and the newly formed upper leaves were collectedfor Southern blot analysis 22 to 25 days following inoculation. To studythe local and systemic movement of the virus expressing greenfluorescent protein [GFP; Chalfie et al, Science, 263:802-805, 1994)],bottom leaves of four-week old seedlings (10 plants per construct) wereinoculated. Inoculated and upper non-inoculated leaves were observed atthree day intervals for fifteen days under a fluorescence microscope forthe detection of fluorescence emitted by GFP. In all experiments thatinvolved plants, wild type B component DNA, which is essential forsystemic spread and symptom development, was included.

3. Southern Blotting

Total DNA was isolated from protoplasts (Mettler et al, Plant Mol. Biol.Rep., 5:346-349, 1987) and plants (Dellaporta et al, Plant Mol. Biol.Rep., 1:19-21, 1983) and electrophoresed in 1% agarose gels (withoutethidium bromide) and transferred to Hybond nylon membranes (Amersham,Arlington Heights, Ill.) using the standard protocols (Sambrook et al,Molecular Cloning: A laboratory manual., Cold Spring harbor laboratorypress. Cold Spring harbor, N.Y., 1989). Hybridization reactions wereperformed using a randomly primed 32P-labeled A component specific probe(the 900 bp Al1 II-Pst I fragment containing ORFs AC1, AC2, and AC3).The amount of viral as and daDNA (super coiled, linear, open circular,and dimeric forms) was quantitated by exposing the Southern blots tostorage phosphor screen plates and counting on a PhosphorImager(molecular Dynamics, Sunnyvale, Calif.). The ssDNA form was confirmed byits susceptibility to S1 and mungbean nucleases (Padidam et al,Virology, 224:390-404, 1996). In the absence of ethidium bromide, thesuper coiled viral DNA form runs ahead of the ssDNA form.

4. Immunoprecipitation and Western Blotting

For immunoprecipitation reactions, protoplasts infected with the virus Acomponent expressing CP66:6G:g5 protein tagged with Flag epitope(FCP66:6G:g5, Table 1) were lysed with a hand held polytron in NP40buffer 50 mM Tris-HCl (pH 7.5), 1% NP40, with 0.15, 0.25, 0.50, 0.75, or1.0 M NaCl} or RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1%NP40, 0.5% DOC, 0.1% SDS] containing a cocktail of protease inhibitors(Boehringer Mannheim, Indianapolis, Ind.). Cell debris was removed bycentrifugation at 4° C. for 10 min at 15,000×g. Lysates wereimmunoprecipitated with anti-Flag monoclonal M2 antibody covalentlylinked to agarose (Sigma, St. Louis, Mo.). Immune complexes were washedfour times with NP40 or RIPA buffer and once with Tris-buffered saline[50 mM Tris-HCl (pH 7.5), 150 mM NaCl]. Half of each sample was heatedin Laemmli sample buffer, fractionated by SDS-PAGE (13% acrylamide), andtransferred to PVDF membrane (Schleicher & Schuell, Keene, N.H.).Immunoprecipitated protein was visualized with anti-Flag M2 antibodyusing ECL-western blot reagents (Pierce, Rockford, Ill.). The remaininghalf of the immune complex collected by this procedure was used forisolating the viral DNA. Whole cell protein extracts for direct westernblotting were prepared by boiling the protoplast pellets with equalvolume of 2×Laemmli sample buffer.

5. Immunofluorescence

Protoplasts transfected with viral constructs were cultured on chamberslides (Nalge Nunc, Rochester, N.Y.) for 48 h, fixed with 3%paraformaldehyde in PBSEM [50 mM phosphate (pH 6.95), 150 mM NaCl, 5 mMEGTA, 5 mM MgSO4] for 30 min, and permeabilized with 100% methanol at−20° C. for 10 min. The cells were washed two times with PBSEMcontaining 0.5% Tween 20 for 30 min. CP66:6G:g5 protein tagged with Stagepitope (CP66:Stag:6G:g5, Table 1) was detected with the S-proteincoupled to FITC (Novagen, Madison, Wis.). The fifteen amino acid longStag peptide was inserted after Arg66 of the CP to construct theCP66:Stag:6G:g5 protein. Flag epitope-tagged BV1, T7 epitope-tagged BC1,CP and β-glucuronidase (GUS) (Table 1) were detected with anti-Flag M2antibody (Sigma, St. Louis, Mo.), anti-T7 tag antibody (Novagen,Madison, Wis.), anti-CP antisera (Padidam et al, Virology, 224:390-404,1996), and anti-GUS antisera (5′-3′, Boulder, Colo.) diluted 1:100 inPBS, respectively. After incubation in primary antibody for 1 h at 30°C., the cells were washed as before and incubated with FITC or rhodamineconjugated IgGs (Pierce, Rockford, Ill.) at a dilution of 1:100. Thecells were mounted in Fluoromount G (Electron Microscopy Sciences, FortWashington, Pa.) and viewed with a Nikon fluorescence microscope orOlympus confocal microscope (for detecting T7 epitope-tagged BC1protein).

6. ToLCV Expressing Gene 5 Protein or CP66:6G:g5 Protein AccumulatesssDNA to Wild Type Levels in Protoplasts

Previous reports work with ToLCV have shown that viral CP and AV2 arenot required for virus replication in protoplasts whereas AV2 isrequired for efficient movement in plants (Padidam et al, Virology,224:390-404, 1996). Coat protein is not essential for systemic movementand symptom development in ToLCV. However, mutations in the CP sequencecaused a marked decrease in ssDNA accumulation in N. bentamiana andtomato plants and in BY2 protoplasts while increasing dsDNA accumulationin protoplasts. Virus that contained mutations in the AV2 plus CPbehaved like AV2 mutants in plants (i.e., poor virus movement and verymild symptoms) and like CP mutants in protoplasts (i.e., decrease inssDNA and increase in dsDNA accumulation).

The present plasmid constructs provide information on the effects ofgene 5 protein (g5p) from E. coli phage M13 (Salstrom et al, J. Mol.Biol., 61:489-501, 1971) on replication of ToLCV. Each of thesemutations are described in Table 1 and FIG. 1. The AV2 and theoverlapping 5′ portion of the CP ORF were replaced with the g5p andassayed its effect on virus replication in protoplasts. In theseexperiments protoplasts were inoculated with wild type (wt) or othermutants, as described below. The modified A component, designatedg5AV2⁻CP⁻, led to accumulation of ssDNA to the same levels as didinfections with wt virus A component (Table 2; FIG. 2, lanes 1 and 3).However, dsDNA accumulation was high (3 to 6 fold higher than wt levels)and similar to accumulation in virus with mutations in CP (Table 2; FIG.2, lanes 2-4). Infection by virus in which the g5 gene was mutated toprevent its translation (g5⁻AV2⁻CP, Table 1) behaved like virusinfections with A component mutants AV2⁻CP⁻ and CP⁻ (Table 2; FIG. 2,lane 4). Since AV2 is required for efficient virus movement in plantsanother construct was made in which g5 was fused to CP at Arg66 withoutaffecting the AV2 ORF (CP66:g5, Table 1). CP66:g5 virus A component alsoled to accumulation of ssDNA, but to lower levels than g5AV2-CP DNA(Table 2; FIG. 2, lane 6). To evaluate whether the N-terminal 66 aminoacids (aa) of CP interfered with the ability of g5p to bind DNA, alinker of six glycine residues was introduced between Arg66 of CP and g5to separate the CP domain from the g5p (CP66:6G:g5). Addition of thelinker restored the ability of the CP66:6G:g5 virus A component toaccumulate ssDNA to levels comparable to those of g5AV2⁻CP⁻ (Table 2;FIG. 2, lane 7). A control construct in which the g5 portion of thefusion protein was not translated (CP66:g5⁻) failed to accumulate ssDNA(Table 2; FIG. 2, lane 8). The ability of virus A component expressingCP66:6G:g5 protein to accumulate ssDNA was not due to N-terminal 66 aaof the CP was suggested by the facts that the virus A componentexpressing g5p alone accumulated ssDNA and the virus A componentsexpressing CP66:6G:BC1 (see below) or CP66:6G:AV2 failed to accumulatessDNA.

TABLE 2 Effect of gene 5 protein on replication and movement of tomatoleaf curl virus in Nicotiana tabacum protoplasts and N. benthamianaplants Protoplast inoculations Virus ssDNA^(a) dsDNA^(a) Wild type^(c)100 100 AV2⁻CP⁻ <1 (0-0.03) 506 (427-584) g5AV2⁻CP⁻ 102 (79-133) 409(349-573) g5⁻AV2⁻CP⁻ 7 (5-12) 384 (210-779) CP⁻ 5 (2-7) 241 (148-369)CP66:g5 17 (8-27) 442 (345-576) CP66:6G:g5 118 (34-234) 517 (133-784)CP66:g5⁻ 9 (3-14) 424 (179-789) Plant inoculations Sym- # of plants ptomVirus inoculated type ssDNA^(b) dsDNA^(b) Wild type^(c) 20 Severe 100100 AV2⁻CP⁻ 10 Very 0.3 (0.05-0.5) 11 (9.6-17) mild^(d) g5AV2⁻CP⁻ 20Very 0.6 (0.1-2.7) 15.2 (6.2-49.2) mild^(d) g5⁻AV2⁻CP⁻ 20 Very 0.1(0.0-0.2) 5.7 (0.0-11.4) mild^(d) CP⁻ 20 Severe^(e) 4.3 (2.6-6.5) 102(65-139) CP66:g5 20 mild 2.2 (0.8-4.2) 30.6 (15.3- 55.1) CP66:6G:g5 30Very 0.9 (0.4-1.7) 10.9 (5.5-14.7) mild^(d) cP66:g5⁻ 20 Severe^(e) 4.0(1.8-6.1) 139.7 (56.0- 197.7) ^(a)The values represent the averageamount (range) of single-stranded (ss) and double-stranded (ds) Acomponent DNA in five independent protoplast transfections per mutant.Protoplasts (˜10⁶) were transfected with 2 μg of A component DNA and 40μg of herring sperm DNA. Viral DNA was quantitated on Southern blotsusing the “phosphorImager” from Molecular Dynamics. ^(b)The values areaverage (range) amounts of viral DNA in twelve inoculated plants pervirus construct except for AV2⁻CP⁻for which the values are averages offour plants. Each plant was inoculated with 0.5 μg of A and 0.5 μg ofwild type B component DNA, which is essential for viral movement andsymptom development. ^(c)The amount of viral DNA in protoplasts andplants inoculated with the wild type viral DNA were assigned a value of100. ^(d)Many plants did not show symptoms. ^(e)Severe symptoms like inplants inoculated with the wild type virus but without intensechlorosis.

Geminiviruses replicate in the nucleus (Accotto et al, Virology,195:257-259, 1993; Nagar et al, Plant Cell, 7:705-719, 1995), so it islikely that in order to cause the accumulation of ssDNA the CP66:6G:g5and g5 proteins must be present in the nucleus. To immunolocalize theCP66:6G:g5 fusion protein in protoplasts, the Stag epitope was insertedbetween Arg66 of the CP and the glycine linker (CP66:Stag:6G:g5, Table1). At 48 h after infection protoplasts were fixed and subjected toreactions with S-protein coupled to FITC. The CP66:Stag:6G:g5 protein aswell as the wt CP (detected with anti-CP antisera) were localized to thenucleus (FIGS. 3A and 3B). When GUS protein was produced as a fusionprotein with the N-terminal 66 aa of CP (CP66:GUS), the GUS (detectedwith anti-GUS antisera) was also localized to the nucleus (FIG. 3C).This indicated that the N-terminal 66 aa of the CP contained a nuclearlocalization signal.

g5p contains a nuclear localization signal as shown by fusing g5sequence to the sequence coding for GUS at the N-terminus. The g5:GUSfusion protein (expressed in g5:GUSAV2⁻CP⁻ virus A component, Table 1)and unfused GUS protein (expressed in GUSAV2⁻CP⁻virus A component,Table 1) remained in the cytoplasm (FIGS. 3D and 3E), indicating thatg5p has no nuclear localization signal. The g5p most likely entered thenucleus in a passive manner based on its size (9.7 kDa) which is smallerthan the permeability barrier of the nuclear envelop (Dingwall et al,Ann. Rev. Cell Biol., 2:367-390, 1986).

7. Movement of ToLCV Expressing CP66:6G:g5 Protein is Impaired in PlantsN. benthamiana plants were inoculated with selected virus constructs todetermine the effect of g5p on virus spread: in these studies Bcomponent DNA was coinoculated with A component onto N. benthamianaseedlings. As expected, plants inoculated with A component mutantsAV2⁻CP⁻, g5AV2⁻CP⁻, or g5⁻AV2⁻CP⁻ plus B component showed very mild orno symptoms and all inoculated plants accumulated low levels of viralDNA (Table 2). A previously reported ToCLV mutant (Padidam et al,Virology, 224:390-404, 1996) that did not produce CP but produced AV2(CP⁻) developed severe disease symptoms and wt levels of dsDNA onsystemic infections (Table 2). Surprisingly, plants inoculated with thevirus expressing CP66:6G:g5 protein showed very mild or no symptoms eventhough the virus contained an intact AV2 gene (Table 2). These plantsaccumulated low levels of viral DNA similar to plants inoculated withAV2⁻CP⁻virus (Table 2). Plants inoculated with the virus expressingCP66:g5 protein (which accumulated ssDNA to a lower level thanCP66:6G:g5 virus in protoplasts) showed mild symptoms and accumulatedmoderate levels of dsDNA. The impaired movement of the virus expressingg5p was due to possible toxic effects of g5p. No differences inprotoplast viability or in appearance of plant leaves inoculated with wtvirus or virus expressing g5p were detected that might suggest toxicityof g5p.

The cell to cell and long distance movement of ToLCV expressingCP66:6G:g5 protein was examined by utilizing green fluorescent protein(GFP) as a visible marker for virus movement. Plants were inoculatedwith A component DNA expressing GFP in place of AV2 and CP (GFPAV2^(−CP)⁻) alone, or coinoculated with A component DNA of the wt, CP66:6G:g5, orCP66:g5⁻ construct. GFPAV2⁻CP⁻ virus was expected to move inefficientlyin plants as it does not encode AV2; it was expected to move efficientlywhen complemented by another virus encoding AV2. GFP could not bedetected in plants by 3 d post inoculation, but it was present oninoculated and upper leaves by day 6 in the majority of the plantsinoculated with GFPAV2⁻CP⁻ plus wt A component, or GFPAV2⁻CP⁻ plusCP66:g5⁻viruses (FIG. 3H, 3I; only data on plants inoculated withGFPAV2⁻CP⁻ plus CP66:g5⁻ viruses is shown). The virus expressing GFPcontinued to spread to upper and newly emerging leaves in these plants(FIG. 3J, 3K). GFP was observed in veins, mesophyll and epidermal cells,and was present in large areas of the leaf in plants inoculated withGFPAV2−CP− plus CP66:g5− viruses. In contrast, GFP was restricted tosmall spots on the inoculated leaves of most of the plants inoculatedwith GFPAV2⁻CP⁻, or GFPAV2⁻CP⁻ plus CP66:6G:g5 viruses (FIG. 3L, 3M;only data on plants inoculated with GFPAV2⁻CP⁻ plus CP66:6G:g5 virusesis shown). These plants also showed GFP staining in some adjacent andnewly emerging leaves, but mostly restricted to veins (FIG. 3N, 30, 3P).These results indicated that expressing the g5p in place of CP hasdecreased the efficiency of the virus systemic movement.

8. In vivo Binding of CP66:6G:g5 Protein to Viral DNA

The accumulation of viral sSDNA in protoplasts inoculated with virus Acomponent expressing g5p or CP66:6G:g5 protein indicated that g5p bindsto ssDNA. In verification, protoplasts were inoculated with virus Acomponent expressing Flag epitope-tagged CP66:6G:g5 protein(FCP66:6G:g5, Table 1) and immunoprecipitated the Flag epitope-taggedCP66:6G:g5 protein using anti-Flag antibody and characterized the viralDNA that coimmunoprecipitated with the CP66:6G:g5 protein by Southernblotting. The immunoprecipitations were performed under different salt(1% NP40 buffer with 0.15 to 1.0 M NaCl) conditions and in the presenceof 0.1 SDS, 0.5% DOC and 1% NP40 detergents (RIPA buffer) to assay theaffinity of binding. Flag epitope-tagged CP66:6G:g5 protein wasimmunoprecipitated in all the buffer conditions tested; the amount ofprotein immunoprecipitated increased with the increase in saltconcentration. (FIG. 4A). The amount of coimmunoprecipitated ssDNAincreased up to 0.5 M salt and decreased at higher concentrations (FIG.4B), indicating the g5p-ssDNA complex was destabilized in buffer thatcontained 1 M salt. Immunoprecipitation in RIPA buffer also resulted inreduced amount of precipitated DNA (FIG. 4B). These results showed thatg5p bound to viral ssDNA and 1 M salt (in NP40 buffer) dissociated g5pfrom viral DNA.

9. Role of BV1 and BC1 Movement Proteins in Spread of ToLCV

Together, the above results indicate that CP66:6G:g5 protein islocalized to the nucleus and binds stably to ToLCV virus DNA in vivo,and ToLCV expressing CP66:6G:g5 does not move efficiently in plants. Theinefficient movement of ToLCV expressing CP66:6G:g5 protein may be dueto interference of g5p with the function of BV1 or BC1 movement proteinsof ToLCV. In squash leaf curl virus (SLCV), BV1 (referred to as BR1 inSLCV) protein, but not BC1 (referred to as BL1 in SLCV), binds to ssDNAin vitro (Pascal et al, Plant Cell, 6:995-1006, 1994) . BV1 and BC1 ofSLCV interact with each other in a cooperative manner; in protoplastsBV1 localizes to the nucleus in the absence of BC1 but localizes to thecell periphery in the presence of BC1 (Sanderfoot et al, Plant Physiol.,110:23-33, 1996; Sanderfoot et al, Plant Cell, 7:1185-1194, 1995). BothBV1 and BC1 are required for the systemic spread and symptom developmentof ToLCV (Padidam et al, Virology, 224:390-404, 1996). To determine ifBV1 and BC1 of ToLCV have similar functions as BV1 and BC1 of SLCV, BV1and BC1 of ToLCV were immunolocalized and examined for their ability tocomplement viral ssDNA accumulation of CP mutants. For these experimentsBV1 and BC1 genes were fused to sequences coding for Flag epitope tagand T7 epitope tag, respectively, and inserted in place of AV2 and CP inthe A component (FBV1AV2⁻CP⁻ and TBC1AV2⁻CP⁻, Table 1). In protoplastsinoculated with FBV1AV2⁻CP⁻ construct, BV1 protein accumulated in thenucleus (detected using anti-Flag antibody, FIG. 3F) while inprotoplasts inoculated with TBC1AV2⁻CP⁻, the BC1 protein was localizedto the cell periphery (detected using anti-T7 tag antibody, FIG. 3G)Expression of BV1 protein in place of AV2 and CP protein (BV1AV2⁻CP⁻)also led to the accumulation of ssDNA of the A component (Table 3; FIG.2, lane 9). The binding affinity of BV1 protein tagged with Flag epitopeto viral DNA in protoplasts inoculated with FBV1AV2⁻CP⁻ DNA wasdetermined by immunoprecipitation reactions similar to those describedin FIG. 4. The binding affinity of BV1 protein to viral ssDNA wassimilar to the binding affinity of CP66:6G:g5 protein to viral DNA. Incontrast to results obtained with the A component DNA expressing BV1, Acomponent DNA expressing BC1 protein in place of AV2 and CP (BC1AV2⁻CP⁻)did not accumulate ssDNA (Table 3; FIG. 2, lane 10). Since BC1 proteinwas localized to the cell periphery, BC1 was fused to N-terminal 66 aaof the CP (CP66:6G:BC1) to direct it to the nucleus. Virus A componentDNA expressing the CP66:6G:BC1 protein also did not accumulate ssDNA(Table 3; FIG. 2, lane 11) showing that BC1 movement protein may notbind to viral ssDNA or the binding affinity was not sufficiently strongenough to result in the accumulation of ssDNA. These results show thatBV1 is localized to the nucleus in the absence of BC1, and BV1 binds toviral ssDNA in vivo.

TABLE 3 Complementation by BV1 and BC1 movement proteins for theaccumulation of tomato leaf curl virus ssDNA in protoplasts^(a) Acomponent B component ssDNA dsDNA Wild type none 100 100 BV1AV2⁻CP⁻ none86 (50-121) 230 (119-195) FB1AV2⁻CP⁻ none 33 (25-54) 47 (40-58)BC1AV2⁻CP⁻ none 2 (1-3) 224 (162-288) Cp66:6G:BC1 none 5 (1-10) 214(180-267) Wild type Wild type 57 (37-78) 61 (42-81) Wild type BC1⁻ 48(38-58) 50 (40-60) AV2⁻CP⁻ Wild type 2.4 (1.2-3.6) 131 (76-187) AV2⁻CP⁻BC1⁻ 2.7 (1.5-4.0) 135 (82-188) CP⁻ Wild type 2.5 (1.6-3.3) 100 (78-121)CP⁻ BC1⁻ 2.9 (2.1-3.7) 106 (98-113) ^(a)Protoplasts were transfectedwith 2 μg of A component DNA with or without 10 μg of B component DNA.Viral single-stranded (ss) and double-stranded (ds) DNA was quantitatedon Southern blots using “phoshorImager” and the values represent theaverage amount (range) of viral DNA in two to five independenttransfections.

In plants inoculated with ToLCV A component containing CP66:6G:g5 geneplus wt B component the expression of CP66:6G:g5 protein is controlledby the relatively strong CP promoter. The CP66:6G:g5 protein producedfrom the A component may out-compete with the BV1 protein (expressedfrom the B component) for DNA binding if the amount of BV1 made underits own promoter is relatively low. We conducted an experiment todetermine if BV1, expressed under its own promoter on the B component,can lead to accumulation of ssDNA. Note that BV1 led to accumulation ofssDNA when expressed in place of CP on A component (Table 3). However,very little viral ssDNA accumulated in protoplasts coinoculated with Acomponent DNA with mutations in CP (CP⁻) plus wt B component DNA (i.e.,expressing both BV1 and BC1) or B component with a mutation in BC1(BC1⁻; i.e, expressing only BV1) (Table 3; FIG. 2, lanes 12-15). Thefailure of BV1 to cause accumulation of ssDNA when expressed from the Bcomponent appeared to be due to low levels of BV1 protein being made; noBV1 protein was detected in protoplasts coinoculated with A componentDNA and B component DNA expressing Flag epitope-tagged BV1 byimmunolocalization and western blotting procedures. These results showthat the B component promoter driving the expression of BV1 is not asstrong as when the gene was expressed from the CP promoter on the Acomponent.

10. Discussion of Examples 1-9

A non-specific ssDNA binding protein (g5) was expressed in place of CPand was monitored for the accumulation of ssDNA to determine if it couldserve as a substitute for CP in Geminivirus. The g5p from E. coli phageM13 was chosen because of its small size (9.7 kDa) and lack of anyenzymatic function in DNA replication. The role of g5p in replication ofM13 and other filamentous phages has been extensively studied (Raschedet al, Microbiol. Rev., 50:401-427, 1986) and its structure has beendetermined (Skinner et al, Proc. Natl. Acad. Sci. USA, 91:2071-2075,1994). Gene 5 protein binds newly formed viral ssDNA tightly,cooperatively, and in a sequence independent manner, and protects itfrom degradation by E. coli nucleases.

It is shown that g5p can bind to ToLCV ssDNA in plant cells and ToLCVexpressing g5p or g5p fused to N-terminal 66 aa of the CP accumulatedssDNA to wt levels. The binding of g5p to viral ssDNA in vivo wassimilar to the binding of g5p to M13 ssDNA in vitro (Anderson et al,Biochemistry, 14:907-917, 1975). Though g5p compensated for the lack ofCP by causing an increase in accumulation of ssDNA of ToLCV, it did notreduce the amount of dsDNA to wt levels. BV1 movement protein (whenexpressed in place of CP) also behaved like g5p in that it did notdown-regulate the dsDNA to wt levels. If CP regulates the levels of ssand dsDNA by depleting the ssDNA available for conversion to dsDNA,expression of g5p or BV1 could be expected to result in normal amountsof dsDNA. The fact that it did not suggests that CP may have a directrole in regulating virus replication, possibly by inhibitingminus-strand synthesis or by regulating gene expression. The CP ofalfalfa mosaic virus (A1MV), a virus with a ssRNA(+) genome, has beenshown to play a direct role in regulation of plus- and minus-strand RNAsynthesis. The A1MV CP was found in tight association with the viral RNApolymerase and inhibited minus-strand synthesis while stimulatingplus-strand synthesis. Recent results on SLCV suggests that CP acts tosignal the switch from viral dsDNA replication to ssDNA replication, orto sequester virion ssDNA from replication pool without fullyencapsidating it. Purification of geminivirus replication complexes isneeded to directly assess the role of CP in replication.

Plants infected with virus that encodes CP66:6G:g5 protein show verymild symptoms and accumulate low levels of viral DNA when infectedprotoplasts accumulated high levels of viral DNA. This occurs because bybinding to viral ssDNA, g5p affects virus movement by interfering withthe function of BV1 movement protein. BV1 of ToLCV was localized to thenucleus in infected protoplasts and bound to viral ssDNA in vivo;whereas BC1 was localized to the cell periphery and did not complementviral ssDNA accumulation even when it was directed to the nucleus as afusion to the nuclear localizing signal of CP. Recent studies on therole of BV1 and BC1 in SLCV movement have shown that BV1 localizes tothe nucleus, binds to ssDNA in vitro, and functions as a nuclear shuttleprotein. BC1 of SLCV is localized to the cell periphery in protoplastsand is associated with endoplasmic reticulum-derived tubules indeveloping phloem cells of systemically infected pumpkin seedlings.Based on these results, a model for SLCV was proposed in which BC1containing tubules serve as a conduit for the transport of BV1, and itsassociated viral ssDNA, from one cell to another (Ward et al, J. Virol.,71:3726-33, 1997). Studies on TGMV have shown that BV1 interacts withviral ssDNA in vivo and BV1 and BC1 have distinct and essential roles incell to cell movement as well as systemic movement (Jeffrey et al,Virology., 223:208-218, 1996). ToLCV employs a similar strategy inmoving from cell to cell. The poor movement of ToLCV that producesCP66:6g:g5 protein is due to reduced binding of BV1 to viral ssDNA. Itshould be noted that BV1 did not lead to accumulation of ssDNA of Acomponent that lacked CP when BV1 was expressed under its own promoterfrom the B component. In plants coinoculated with A component producingCP66:6G:g5 plus A component producing GFP, GFP staining was mostlyrestricted to small areas, both on inoculated and systemically infectedleaves, showing an over all reduction in the efficiency of viralmovement than specific interference with cell to cell spread or longdistance movement.

The interference with the ToLCV movement due to binding of g5p to viralssDNA indicates that in this virus ssDNA moves from cell to cell. Theseresults also indicate that expression of g5p in transgenic plantsprovides a novel way of controlling geminiviruses and that suchresistance is effective against all geminiviruses.

In summary, to determine whether the gene 5 protein (g5p), a ssDNAbinding protein from Escherichia coli phage M13, could restore theaccumulation of ssDNA, ToLCV that lacked the CP gene was modified toexpress g5p or g5p fused to the N-terminal 66 amino acids of the CP(CP66:6G:g5). The modified viruses led to accumulation of wild typelevels of ssDNA and high levels of dsDNA. The accumulation of ssDNA wasdue to stable binding of g5p to the viral ssDNA. The high levels ofdsDNA accumulation during infections of the modified viruses indicatedsuggested a direct role for CP in viral DNA replication. ToLCV thatproduced CP66:6G:g5 protein did not spread efficiently in Nicotianabenthamiana plants and inoculated plants developed only very mildsymptoms. In infected protoplasts CP66:6G:g5 protein was immunolocalizedto nuclei; this indicates that the fusion protein interferes with thefunction of BV1 movement protein and thereby prevents spread of theinfection.

The foregoing specification, including the specific embodiments andexamples, is intended to be illustrative of the present invention and isnot to be taken as limiting. Numerous other variations and modificationscan be effected without departing from the true spirit and scope of theinvention.

1. A method for producing in a plant resistance to a single stranded DNA(ssDNA) virus of the Geminivirus family comprising introducing a gene 5ssDNA-binding protein of Coliphage M13 into said plant, therebyproducing resistance to said ssDNA virus in said plant.
 2. The method ofclaim 1 wherein said Coliphage M13 gene 5 protein has the amino acidresidue sequence of SEQ ID NO
 1. 3. The method of claim 1 wherein saidintroducing comprises preparing a transgenic plant containing a genewhich expresses said ssDNA-binding protein.
 4. The method of claim 3wherein said gene comprises a nucleotide sequence shown in SEQ ID NOs 2or
 3. 5. The method of claim 1 wherein said introducing comprisescontacting said plant with a composition containing an expression vectorcapable of expressing said ssDNA-binding protein.
 6. The method of claim5 wherein said expression vector comprises a nucleotide sequence shownin SEQ ID NOs 2 or
 3. 7. The method of claim 5 wherein said contactingcomprises biolistic gene transfer or direct DNA uptake into protoplast.8. The method of claim 5 wherein said contacting comprises infection ofsaid plant with a carrier vector.
 9. The method of claim 8 wherein saidcarrier vector is an Agrobacterium vector.
 10. The method of claim 5wherein said expression vector is present in a virus particle thatinfects said plant and expresses said ssDNA-binding coat protein. 11.The method of claim 1 wherein said plant is selected from the groupconsisting of Abutilon, Acalypha, apple, Ageratum, Althea rosea,Asystasia, Bajra, banana, barley, beans, beet, Blackgram, Bromus,Cassava, chickpea, Chilllies, Chloris, clover, coconut, coffee, cotton,cowpea, Croton, cucumber, Digitaria, Dolichos, eggplant, Eupatorium,Euphorbia, fababean, honeysuckle, horsegram, Jatropha, Leonurus,limabean, Lupin, Macroptilium, Macrotyloma, maize, melon, millet,mungbean, oat, okra, Panicum, papaya, Paspalum, peanut, pea, pepper,pigeon pea, pineapple, Phaseolus, potato, Pseuderanthemum, pumpkin,Rhynchosia, rice, Serrano, Sida, sorghum, soybean, squash, sugarcane,sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon,wheat and Wissadula.
 12. The method of claim 1 wherein said Geminivirusis selected from the group consisting of Mastrevirus, Curtovirus andBegomovirus genera.
 13. A method for producing geminivirus resistance ina plant comprising introducing into said plant a gene capable ofexpressing Coliphage M13 gene 5 protein in said plant, thereby producingresistance to said geminivirus in said plant.
 14. A DNA expressionvector comprising a nucleotide sequence that encodes a gene 5ssDNA-binding protein of Coliphage M13, wherein said vector is capableof expressing said protein in plants.
 15. The DNA expression vector ofclaim 14 wherein said Coliphage M13 gene 5 protein has the amino acidresidue sequence of SEQ ID NO
 1. 16. The DNA expression vector of claim14 wherein said nucleotide sequence comprises a nucleotide sequenceshown in SEQ ID NOs 2 or
 3. 17. The DNA expression vector of claim 14wherein said vector is a carrier vector.
 18. The DNA expression vectorof claim 17 wherein said carrier vector is an Agrobacterium vector. 19.The DNA expression vector of claim 14 wherein said plant is selectedfrom the group consisting of Abutilon, Acalypha, apple, Ageratum, Althearosea, Asystasia, Bajra, banana, barley, beans, beet, Blackgram, Bromus,Cassava, chickpea, Chilllies, Chloris, clover, coconut, coffee, cotton,cowpea, Croton, cucumber, Digitaria, Dolichos, eggplant, Eupatorium,Euphorbia, fababean, honeysuckle, horsegram, Jatropha, Leonurus,limabean, Lupin, Macroptilium, Macrotyloma, maize, melon, millet,mungbean, oat, okra, Panicum, papaya, Paspalum, peanut, pea, pepper,pigeon pea, pineapple, Phaseolus, potato, Pseuderanthemum, pumpkin,Rhynchosia, rice, Serrano, Sida, sorghum, soybean, squash, sugarcane,sugarbeet, sunflower, sweet potato, tea, tomato, tobacco, watermelon,wheat and Wissadula.
 20. A composition for producing resistance to assDNA virus of the Geminivirus family that infects plants comprising aDNA expression vector comprising a nucleotide sequence that encodes agene 5 ssDNA-binding protein of Coliphage M13, wherein said vectorexpresses said protein in said plant.
 21. The composition of claim 20wherein said Coliphage M13 gene 5 protein has the amino acid residuesequence of SEQ ID NO
 1. 22. The composition of claim 20 wherein saidnucleotide sequence comprises a nucleotide sequence shown in SEQ ID NOs2 or
 3. 23. The composition of claim 20 wherein said DNA expressionvector is a carrier vector.
 24. The composition of claim 23 wherein saidcarrier vector is an Agrobacterium vector.
 25. A transgenic plantcontaining a DNA expression vector comprising a nucleotide sequence thatencodes a gene 5 ssDNA-binding protein of Coliphage M13, wherein saidvector expresses said protein in said plant.
 26. The transgenic plant ofclaim 25 wherein said DNA expression vector is the vector of claim 14.27. The transgenic plant of claim 25 wherein said Coliphage M13 gene 5protein has the amino acid residue sequence of SEQ ID NO
 1. 28. Thetransgenic plant of claim 25 wherein said nucleotide sequence comprisesa nucleotide sequence shown in SEQ ID NOs 2 or 3.